Biofilms are biological films that develop and persist at interfaces in aqueous environments (Geesey, et al., Can. J. Microbiol. 32. 1733–6, 1977; 1994; Boivin and Costerton, Elsevier Appl. Sci., London, 53–62, 1991; Khoury, et al., ASAIO, 38, M174–178, 1992; Costerton, et al., J. Bacteriol., 176, 2137–2142, 1994), especially along the inner walls of conduit material in industrial facilities, in household plumbing systems, on medical implants, or as foci of chronic infections. These biological films are composed of microorganisms embedded in an organic gelatinous structure composed of one or more matrix polymers which are secreted by the resident microorganisms. Biofilms can develop into macroscopic structures several millimeters or centimeters in thickness and can cover large surface areas. These biological formations can play a role in restricting or entirely blocking flow in plumbing systems and often decrease the life of materials through corrosive action mediated by the embedded bacteria. Biofilms are also capable of trapping nutrients and particulates that can contribute to their enhanced development and stability.
The involvement of extracellular polymers in bacterial biofilms has been documented for both aquatic (Jones, et al., J. Bacteriol., 99, 316–325, 1969; and marine bacteria (Floodgate, Memorie dell'Instituto italiano di idrobiologica Dott. Carco di Marchi 29 (suppl.), 309–323 1972), and the association of exopolysaccharides with attached bacteria has been demonstrated using electron microscopy (Geesey, et al., supra; Dempsey, Marine Biol. 61, 305–315, 1981) and light microscopy (Zobell, J. Bacteriol. 46, 39–56, 1943. The presence of such exopolysaccharides is believed to be involved in the development of the microbial biofilm (Allison and Sutherland, J. Microbiol. Methods, 2, 93–99, 1987). Analysis of biofilm bacteria isolated from freshwater and marine environments has shown that the polymers they produce are composed largely of acidic polysaccharides. The control and removal of biofilm material from pipe and conduit surfaces has historically been carried out by the addition of corrosive chemicals such as chlorine or strong alkali solutions or through mechanical means. Such treatments are generally harsh to both the plumbing systems and the environment, and have been necessary due to the recalcitrant nature of biofilms within those systems. The resistance to treatment by biocides has been due in large measure to the protective character of intact biofilm matrix polymers (Srinivasan, et al., Biotech. Bioeng., 46, 553–560, 1995.
Modern methods of direct observation of living biofilms have established the very complicated structural architecture (Costerton, et al., Ann., Rev. Microbiol., 49, 711–745, 1995) of these sessile microbial populations. The overriding structural element responsible for development and maintenance of this biofilm architecture is the matrix polymer that is produced by the resident microorganisms. That populations of associated bacteria could produce structures as complex as are found in biofilms suggested the operation of a cell-cell signaling mechanism.
Prior to 1981, microbiologists had generally assumed that bacteria had neither the requirement nor the capability of producing cell-cell signaling molecules. In 1981, it was shown by Eberhard, et al. Biochemistry, 20, 2444–2499, 1981, that the bacterium Photobacterium fischeri produces a compound 3-oxo-N-(tetrahydro-2-oxo-3-furanyl) hexanamide, also known as vibrio (photobacterium) autoinducer (VAI), which is associated with bacterial luminescence under conditions of high cell density. The cell membrane of P. fischeri was shown to be permeable to VAI by Kaplan and Greenberg in 1985 (J. Bacteriol., 163, 1210–1214, 1985). At low bacterial cell densities in broth medium, VAI passively diffuses out of the cells along a concentration gradient, where it accumulates in the surrounding medium. At high cell densities the concentration of VAI outside the cells is equivalent to the concentration of VAI inside the cells. Under such conditions VAI was shown to initiate transcription of luminescence genes. Using such a system, bacteria are able to monitor their own population density and regulate the activity of specific genes at the population level.
For several years it was presumed that the autoinducer involved in bacterial luminescence was unique to the few bacteria that produce light in the marine environment. Then, in 1992, the terrestrial bacterium Erwinia carotovora was shown to use an autoinducer system to regulate the production of the β-lactam antibiotic carbapenem (Bainton, et al., Biochem J., 288, 297–1004, 1992b). The molecule found to be responsible for autoinduction of carbapenem was shown to be an acylated homoserine lactone (HSL), a member of the same class of molecule responsible for autoinduction in bioluminescence. This finding led to a general search for HSLs in a wide range of bacteria. To affect the search, a bioluminescence sensor system was developed and used to screen for HSL production in the spent supernatant liquids of a number of bacterial cultures. Many different organisms were shown by the screening to produce HSLs. These included: Pseudomonas aeruginosa, Serratia marcescens, Erwinia herbicola, Citrobacter freundii, Enterobacter agglomerans and Proteus mirabilis (Brainton, et al., Gene. 116, 87–91, 1992a; Swift, et al., Mol. Microbiol., 10, 511–520, 1993). More recently, the list has grown to include Erwinia stewartii (Beck, J. Bacteriol, 177, 5000–5008, 1993), Yersinia enterocolitica (Throup, et al., Mol. Microbiol., 17, 345–356, 1995), Agrobacterium tumefaciens (Zhang, et al., Nature, 362, 446–448, 1993), Chromobacterium violaceum (Winston, et al., Proc. Natl. Acad. Sci., USA, 92, 9427–9431, 1995), Rhizobium leguminosarium (Schripsema, et al., J. Bacteriol, 178, 366–371 1996) and others. Today it is generally assumed that all enteric bacteria, and the gram negative bacteria generally, are capable of cell density regulation using HSL autoinducers.
In 1993 Gambello, et al. Infect. Immun., 61, 1880–1184, (1993) showed that the α-HSL product of the LasI gene of Pseudomonas aeruginosa controls the production of exotoxin A, and of other virulence factors, in a cell density dependent manner. Since that time, the production of a large number of Pseudomonas virulence factors have been shown to be controlled by α-HSL compounds produced by the LasI and RhlI regulatory systems (Ochsner, et al., Proc. Natl. Acad. Sci., USA 92, 6424–6428, 1995; Winson, et al., supra; Latifi, et al., 1995), in a manner reminiscent of the Lux system. Latifi, et al. Mol. Microbiol, 21, 1173–1146, (1996) have also shown that many stationary phase properties of P. aeruginosa, including those controlled by the stationary phase sigma factor (RpoS), are under the hierarchical control of the LasI and RhlI cell-cell signaling systems.
In all cases, homoserine lactone autoinducers are known to bind to a DNA binding protein homologous to LuxR in Photobacterium fischeri, causing a conformational change in the protein initiating transcriptional activation. This process couples the expression of specific genes to bacterial cell density (Latifi, et al. supra, 1996). Regulation of this type has been called ‘quorum sensing’ because it suggests the requirement for a ‘quorate’ population of bacterial cells prior to activation of the target genes (Fuqua, et al., J. Bacteriol., 176, 269–275, 1994b). Expression of certain of these ‘virulence factors’ has been correlated with bacterial cell density (Finley and Falkow, Microbiol. Rev. 53, 210–230, 1989).
In P. aeruginosa, quorum sensing has been shown to be involved in the regulation of a large number of exoproducts including elastase, alkaline protease, LasA protease, hemolysin, cyanide, pyocyanin and rhamnolipid (Gambello, et al., supra; Latifi, et al., supra; Winson, et al., supra; Ochsner, et al., 1995); but has never before been shown to be involved in biofilm formation. Most of these exoproducts are synthesized and exported maximally as P. aeruginosa enters stationary phase.
The concept of cell signalling and quorum sensing has been studied in the art. See for example U.S. Pat. No. 5,591,872, to Pearson et al; Passador et al, Journal of Bacteriology, pages 5990–6000, October, 1996; PCT WO92/18614 and U.S. Pat. No. 5,593,827.
However, none of these prior publications recognize how these principles may be applied to enable the regulation of biofilms according to this invention.